Application of wheat disease resistance factor TaCBL4 in the prevention and treatment of stem base rot

Abstract

The application belongs to the technical field of genetic engineering, and relates to application of a wheat disease resistance factor TaCBL4 in prevention and treatment of stem base rot. TaCBL4 The application obtains a wheat strain capable of overexpressing a wheat disease resistance factor gene by using an agrobacterium-mediated method, compares the disease incidence and fungal biomass after infection of a stem base rot pathogen of a receptor wheat Fielder, and knows that the wheat disease resistance factor TaCBL4 plays a positive regulation role in the immune response of wheat against stem base rot. Based on this, the wheat disease resistance factor TaCBL4 provided by the application can be used to create wheat germplasm material resistant to stem base rot, and plays an important role in prevention and treatment of wheat stem base rot.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and relates to application of a wheat disease resistance factor TaCBL4 in prevention and treatment of crown rot. BACKGROUND

[0002] Crown rot (FCR) is a kind of soil-borne disease caused by a variety of pathogenic fungi, and is a kind of disease in which root system and stem base are rotted by single or complex infection of a variety of pathogenic fungi. The pathogenic fungi of the crown rot mainly include Pseudocercospora graminicola (Cooke) Jenkins, Fusarium graminearum (Schwein) S. Ito et al., Fusarium culmorum (Owen) W. Gams, Fusarium cerealis (Cavara) Bres., Fusarium asiaticum (Nishikado et al.) Watanabe, Fusarium avenaceum (Corda) Sacc., Fusarium oxysporum (Schlechtendahl) S. Fender, and Fusarium equiseti (Corda) Sacc. Fusarium Fusarium pseudograminearum F. graminearum F. F. culmorum F. asiaticum F. avenaceum F. F. oxysporum F. equiseti The crown rot is a soil-borne disease of wheat, which not only causes yield loss, but also causes quality decline of the wheat, and seriously threatens the safety of wheat production. Therefore, it is an important means to prevent and control the crown rot of wheat to explore the resistance genes of the wheat and improve the disease resistance of the wheat.

[0003] Pseudocercospora graminicola overwinters on crop residues in mycelial or sporodochial form, and forms asexual macroconidia after germination to further infect the wheat. Pseudocercospora graminicola can survive in the residues for up to 3 years, and the planting mode of no-tillage and stubble retention to maintain soil moisture and nutrients is the main reason for the increase of the incidence of the crown rot in many areas. As a soil-borne disease, the pathogenic fungi of the crown rot infect the root system of the wheat, and cause the disease in the stem base and leaf sheath of the wheat. For the seeds that have been infected or after the seeds are sown, Pseudocercospora graminicola will inhibit the growth of the wheat embryo sheath, cause the seed to rot, and reduce the emergence rate. The wheat plants infected by the pathogenic fungi have deep brown stripes or spots in the stem base, and the stripes or spots spread upward along the leaf sheath, stem node and stem. If the temperature and humidity are suitable, a pink mold layer will appear between the stem nodes. At the mature stage, the wheat plants with serious disease have empty ears or white ears due to the blockage of the vascular bundle by the mycelium, which cannot transport nutrients and water.

[0004] ​​​​​​​During their growth and development, plants are exposed to various biotic and abiotic stresses. Increasing research indicates that stress responses depend on complex interactions between multiple signaling pathways within plant cells. These regulatory networks link plant growth and stress tolerance, thereby mitigating the impact of environmental changes on plants. Therefore, understanding the molecular regulatory networks associated with stress responses and plant development is crucial for improving crop disease resistance and yield. Calcium ion signaling is a key signaling molecule in plant immunity; calcium ions are often considered a "second messenger." When plants are infected by microorganisms, they transiently produce calcium ions. 2+ Changes in calcium concentration trigger a series of immune defense responses, including calcium ion signaling and gene expression regulation. In cells, calcium signals bind to specific calcium-sensing proteins. Currently, four major calcium-sensing proteins are known in the plant kingdom: calmodulin (CaM), calmoneurin B protein (CBL), calcium-dependent protein kinase (CDPK), and calcium ion or calmodulin-dependent protein kinase (CCaMK). These calcium-sensing proteins play important regulatory roles in plants, jointly participating in the plant's response to and transmission of calcium signals. These four calcium-sensing proteins affect calcium at the transcriptional or post-transcriptional levels. 2+ The CBL-CIPK signaling system plays a crucial regulatory role in various physiological processes in plants, including but not limited to responses to biotic and abiotic stresses, regulation of growth and development, nutrient absorption and utilization, and regulation of ROS (reactive oxygen species) signaling. Through precise regulation of these physiological processes, the CBL-CIPK signaling system ensures that plants can maintain normal life activities and adapt to various challenges in complex and changing environments. Therefore, the CBL-CIPK signaling mechanism plays an important role in the entire growth and development process of plants. TaCBL4 belongs to the wheat CBL-CIPK signaling system; its disease resistance and applications are relatively limited and require further in-depth research. Summary of the Invention

[0005] To clarify the role of wheat TaCBL4 in the interaction between wheat and pathogens, and to provide more superior disease-resistant genes, this invention provides the application of wheat disease resistance factor TaCBL4 in the control of stem base rot.

[0006] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the TaCBL4 protein described in this invention is represented by "TaCBL4" in non-italicized font. TaCBL4 Genes in italic font TaCBL4 This indicates that, of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins based on the description in this invention.

[0007] In one aspect, the present application relates to a method for obtaining a plant resistant to stem base rot, comprising: promoting the transcription or translation of a gene in the plant, or promoting the expression level of a TaCBL4 protein in the plant; TaCBL4 promoting the transcription or translation of a gene in the plant, or promoting the expression level of a TaCBL4 protein in the plant;

[0008] the gene encodes the TaCBL4 protein; TaCBL4 the gene encodes the TaCBL4 protein;

[0009] The amino acid sequence of the TaCBL4 protein is shown as SEQ ID NO: 1.

[0010] SEQ ID NO: 1 is specifically as follows:

[0011] MGCAFSSSPSPRKREQRAQGYEEPAVLAAETSFTVNEVEALYELYKKLSFSIFKDGLIHKEEFRLALFRTSRGANLFADRVFDLFDLKRNGVIEFGEFVRSLSIFHPKAPESEKTAFAFKLYDLRGTGYIEKEELREMVVALLDESDLCLSDSAVEEIVHNTFSQADSDGDGRIDPKEWEEFVKQNPASLRNMSLPYLQDITTTFPSFVMHSEVEDYSGISK*.

[0012] In SEQ ID NO: 1, “*” represents a terminator.

[0013] Further, in the method for obtaining a plant resistant to stem base rot provided by the present application, the gene is a gene encoding a TaCBL4 protein. TaCBL4 The CDS sequence of the gene is shown as SEQ ID NO: 2.

[0014] SEQ ID NO: 2 is specifically as follows:

[0015] ATGGGCTGCGCGTTCTCGTCGTCGCCGTCGCCACGGAAGCGTGAGCAGCGCGCCCAAGGGTACGAGGAGCCCGCCGTCCTCGCCGCCGAGACCTCCTTCACGGTGAACGAGGTGGAGGCGCTGTACGAGCTCTACAAGAAGCTCAGCTTCTCCATCTTCAAGGACGGCCTCATCCACAAGGAGGAGTTCCGGCTGGCTCTGTTCAGGACCAGCAGAGGGGCGAACCTGTTCGCGGACAGGGTGTTCGACCTCTTCGATCTCAAGCGCAACGGCGTGATCGAGTTCGGCGAGTTCGTGCGCTCGCTCAGCATCTTCCACCCCAAAGCGCCTGAATCTGAAAAGACCGCGTTTGCATTCAAGCTGTACGATCTGCGGGGGACAGGCTACATCGAGAAAGAAGAGCTCCGGGAGATGGTGGTGGCGCTTCTTGATGAGTCCGACCTATGTCTCTCCGATAGCGCCGTCGAGGAGATTGTCCATAATACGTTCAGTCAAGCAGACTCGGATGGTGATGGCAGGATAGACCCCAAGGAGTGGGAGGAGTTTGTCAAGCAGAACCCAGCGTCGCTGAGGAACATGTCACTGCCCTATCTCCAGGACATTACGACGACATTTCCGAGCTTTGTAATGCATTCCGAAGTCGAAGACTACAGTGGAATCAGCAAATAA.

[0016] Further, in the method for obtaining a plant resistant to stem base rot provided by the present application, the plant is wheat.

[0017] Further, in the method for obtaining a plant resistant to stem base rot provided by the present application, the pathogen of stem base rot is Pseudocercosporella herpotrichoides. Fusarium pseudograminearum .

[0018] Further, in the method for obtaining a plant resistant to stem base rot provided by the present application, a vector over-expressing the gene is constructed, the vector is transformed into Agrobacterium, and the Agrobacterium is used to infect the plant. TaCBL4

[0019] ​Further, in the method for providing a plant resistant to stem base rot provided by the present application, the upstream primer for constructing the vector is shown as SEQ ID NO: 3, and the downstream primer is shown as SEQ ID NO: 4.

[0020] SEQ ID NO: 3 is specifically as follows:

[0021] CAGGTCGACTCTAGAGGATCCATGGGCTGCGCGTTCTCGTC.

[0022] SEQ ID NO: 4 is specifically as follows:

[0023] GAGCTCGGTACCCGGGGATCCTTTGCTGATTCCACTGTAGT.

[0024] In another aspect, the present application relates to TaCBL4 application of the gene in preventing and treating stem base rot, improving the transcription or translation of the gene in a plant, or improving the expression level of the TaCBL4 protein in the plant, and improving the resistance of the plant to stem base rot; TaCBL4 application of the gene in preventing and treating stem base rot, improving the transcription or translation of the gene in a plant, or improving the expression level of the TaCBL4 protein in the plant, and improving the resistance of the plant to stem base rot;

[0025] application of the gene in preventing and treating stem base rot, improving the transcription or translation of the gene in a plant, or improving the expression level of the TaCBL4 protein in the plant, and improving the resistance of the plant to stem base rot; TaCBL4 application of the gene in preventing and treating stem base rot, improving the transcription or translation of the gene in a plant, or improving the expression level of the TaCBL4 protein in the plant, and improving the resistance of the plant to stem base rot;

[0026] The amino acid sequence of the TaCBL4 protein is shown as SEQ ID NO: 1.

[0027] application of the gene in preventing and treating stem base rot provided by the present application, TaCBL4 application of the gene in preventing and treating stem base rot provided by the present application, TaCBL4 The CDS sequence of the gene is shown as SEQ ID NO: 2.

[0028] application of the gene in preventing and treating stem base rot provided by the present application, TaCBL4 application of the gene in preventing and treating stem base rot provided by the present application, the plant is wheat.

[0029] application of the gene in preventing and treating stem base rot provided by the present application, TaCBL4 application of the gene in preventing and treating stem base rot provided by the present application, the pathogenic bacteria of the stem base rot is Pseudocercosporella herpotrichoides Fusarium pseudograminearum .

[0030] Compared with the prior art, the technical scheme provided by the present application at least has the following beneficial effects or advantages:

[0031] The present application proves the feasibility of using the wheat disease resistance factor TaCBL4 to breed wheat varieties resistant to stem base rot. The present application uses the Agrobacterium-mediated method to obtain wheat lines capable of overexpressing the disease resistance factor, and by comparing the disease incidence and fungal biomass of the Fielder receptor wheat after being infected with the physiological race WZ-8A of the false smut fungus, it is known that the wheat disease resistance factor TaCBL4 plays a positive regulatory role in the immune response of wheat against stem base rot. Based on the characteristics of the above-mentioned wheat disease resistance factor TaCBL4, it can be used to create wheat germplasm materials resistant to stem base rot and play a practical role in the prevention and control of wheat stem base rot. The present application gives a preferred method: constructing a recombinant overexpression vector containing the coding gene of the wheat disease resistance factor TaCBL4, and then transforming it into wheat plants to obtain wheat lines capable of overexpressing the disease resistance factor through subculture. The present application provides a technical approach for the cultivation of wheat varieties resistant to stem base rot, and provides a method for the prevention and control of wheat stem base rot, thereby reducing the pathogen resistance and environmental pollution caused by the abuse of pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, a brief introduction will be given below to the drawings to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort.

[0033] Figure 1 For wheat TaCBL4 The schematic diagram of the overexpression recombinant vector of the gene.

[0034] Figure 2 For overexpression TaCBL4 The acquisition result diagram of the transgenic wheat plants of the gene. Wherein, A is the T3 generation TaCBL4 -OE2, TaCBL4 -OE5, TaCBL4 -OE6 molecular detection; B is TaCBL4 The relative expression amount of the gene in the control group (Fielder) and the overexpression line ( TaCBL4 -OE2, TaCBL4 -OE5, TaCBL4 -OE6); C is the phenotype before inoculation of the control group (Fielder) and the overexpression line ( TaCBL4 -OE2, TaCBL4 -OE5, TaCBL4 -OE6).

[0035] TaCBL4 For overexpression Figure 3 The disease resistance verification diagram of the transgenic wheat plants of the gene. Specifically,TaCBL4 Gene overexpression lines (OE1, OE2, OE5, OE6) and control (Fielder) plants' disease incidence and fungal DNA / wheat DNA biomass statistics. TaCBL4 -OE2, TaCBL4 -OE5, TaCBL4 -OE6) and control (Fielder) plants' disease incidence and fungal DNA / wheat DNA biomass statistics. DETAILED DESCRIPTION

[0036] Hereinafter, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. The % in the following examples is the mass percentage unless otherwise specified. The ratio in the following examples is the mass ratio unless otherwise specified.

[0037] Example 1

[0038] This example provides the obtaining of the coding gene of the wheat disease resistance factor TaCBL4.

[0039] The nucleotide sequence of the coding gene of the wheat disease resistance factor TaCBL4 was obtained through the Ensembl Plants (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index) website, and primers for amplifying the full-length of the gene were designed according to the nucleotide sequence, and the primers used were: TaCBL4

[0040] TaCBL4 F: 5'-ATGGGCTGCGCGTTCTCGTCGTCGC-3';

[0041] TaCBL4 R: 5'-TTATTTGCTGATTCCACTGTAGTCT-3'.

[0042] The cDNA of the Fielder wheat plant was used as a template to amplify the gene and perform sequencing, and the CDS sequence of the gene was obtained as shown in SEQ ID NO: 2, and the amino acid sequence encoded thereby was shown as SEQ ID NO: 1. TaCBL4 TaCBL4

[0043] Example 2

[0044] This example provides the construction of transgenic lines (overexpression lines) and the identification of disease resistance.

[0045] According to the ordinary skill of those skilled in the art combined with the prior art, the gene amplified in Example 1 was used to construct transgenic lines (overexpression lines) and identify the disease resistance. TaCBL4 ​​​The full-length sequence of the gene (SEQ ID NO: 2) is constructed into the overexpression vector CUB by a homologous recombination Gateway reaction TaCBL4 The amplified fragment of the full-length gene is constructed into the overexpression vector CUB to form a recombinant overexpression vector TaCBL4 -CUB. The successfully constructed recombinant overexpression vector TaCBL4 -CUB is transfected into Fielder wheat plants by an Agrobacterium (EHA105)-mediated method to obtain transgenic plants capable of overexpressing TaCBL4 the gene.

[0046] The recombinant overexpression vector TaCBL4 -CUB is constructed using the primers:

[0047] TaCBL4 -CUB-F: 5'-CAGGTCGACTCTAGAGGATCCATGGGCTGCGCGTTCTCGTC-3';

[0048] TaCBL4 CUB-R: 5'-GAGCTCGGTACCCGGGGATCCTTTGCTGATTCCACTGTAGT-3'.

[0049] The transgenic plants are subjected to PCR detection and qRT-PCR detection, and positive lines (OE2, OE5 and OE6) are selected according to the detection results, and high-generation wheat lines capable of stably inheriting overexpression TaCBL4 the gene are obtained by subculture. The high-generation wheat lines are inoculated with the physiological race WZ-8A of Pseudocercosporella herpotrichoides, and Fielder wheat plants are used as controls to determine the resistance of the overexpression TaCBL4 gene lines.

[0050] The T3 generation TaCBL4 gene overexpression lines constructed based on the above technical idea ( TaCBL4 -OE2, TaCBL4 -OE5, TaCBL4 -OE6) and the control group (Fielder) wheat plants are inoculated with the physiological race WZ-8A of Pseudocercosporella herpotrichoides after the second leaf unfolds, and the disease conditions of the plants in each group are observed 16 days after inoculation. The inoculation method and resistance evaluation refer to the local standard “DB41 / T 2392-2023 Wheat Resistance to Stem Base Rot Evaluation Technical Specification”.

[0051] TaCBL4 The T3 generation TaCBL4 gene overexpression lines ( Figure 3 -OE5, Figure 3 -OE6) and the control group (Fielder) plants have disease conditions as TaCBL4 shown. From TaCBL4It was found that brown spots were observed at the base of wheat stems in all groups under infection with Fusarium pseudogramensis physiological race WZ-8A. However, TaCBL4 The severity of stem rot in the gene-overexpressing lines was lower than that in the control group.

[0052] Example 3

[0053] This embodiment provides an analysis of the biomass ratio of pathogen DNA to wheat DNA after transgenic lines were inoculated with Fusarium pseudograss physiological race WZ-8A.

[0054] T3 generation cells that tested positive for PCR TaCBL4 Gene overexpression lines ( TaCBL4 -OE2、 TaCBL4 -OE5、 Ta18SrRNA Both the control group (Fielder) and the control group (-OE6) were inoculated with Fusarium pseudobulb physiological race WZ-8A after the second leaf unfolded, and samples were taken for analysis 16 days after inoculation.

[0055] Wheat plant stems, approximately 2 cm long from the base, were cut, wrapped in aluminum foil, and flash-frozen in liquid nitrogen at -80˚C for later use. Total RNA was extracted from wheat leaves using the Trizol (TianGen) method, and first-strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized using the SMART method. qRT-PCR was then used for detection. FpGAPDH Gene expression levels. Wheat tissue DNA was diluted to 4... 2 4 3 4 4 4 5 4 6 4 7 The DNA of *Fusarium graminearum* race WZ-8A was diluted by the same factor as above. Using the diluted DNA as a template, the wheat ribosomal small subunit gene was used. Ta18SrRNA and the gene of glyceraldehyde-3-phosphate dehydrogenase in pathogens FpGAPDH Primers were used to perform qRT-PCR, and a standard curve was plotted between the obtained Cq values ​​and the dilution gradient.

[0056] The DNA from wheat tissues inoculated with Fusarium pseudograecum WZ-8A was diluted to 4... 4 Using wheat as a template, Ta18SrRNA and germs Ta18SrRNA Using primers, qRT-PCR was performed. The obtained Cq values ​​were substituted into the standard curve obtained above to calculate the ratio of pathogen DNA to wheat DNA biomass.

[0057] The qRT-PCR primer sequences are:

[0058] FpGAPDH F: 5'-GTGACGGGTGACGGAGAATT -3';

[0059] FpGAPDH R: 5'-GACACTAATGCGCCCGGTAT-3'.

[0060] Figure 3 F: 5'-GAAGGTCATCATCTCTGCCC-3';

[0061] TaCBL4 R: 5'-GTCTTCTGGGTGGCAGTGTA-3'.

[0062] The quantitative PCR primers need to be detected for the specificity of the amplification product and the amplification efficiency before use, and the amplification efficiency should be ≥ 90%. AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and Bio-Rad CFXManager quantitative PCR instrument (Bio-rad, Hercules, California) were used, and the instructions were referred to, and the DNA of each gene sampling point was used as a template for qRT-PCR. Each gene was repeated 3 times, and the Ct value, average value and standard deviation of each repetition were generated by manual adjustment of the baseline by the quantitative PCR instrument, the experimental data were analyzed by the Delta Delta Ct method, and the biomass ratio of pathogen DNA / wheat DNA was determined.

[0063] Figure 3 The left graph is the schematic diagram of the overexpression transgenic plants inoculated with Pseudocercosporella herpotrichoides showing resistance, and the right graph is the biomass analysis after inoculation for 16 days. TaCBL4 As can be seen from the middle graph, compared with the control plant Fielder, TaCBL4 The fungal biomass of the overexpression transgenic plants is significantly reduced. ​ The fungal biomass of the overexpression transgenic plants is significantly reduced.

[0064] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents the preferred embodiments of the present application. All other embodiments obtained by related deduction and replacement made by those skilled in the art under the condition of the concept of the present application, without making creative labor, belong to the scope of protection of the present application.

Claims

1. A method of obtaining a plant resistant to stem base rot, characterized by, Comprising: overexpressing in said plant TaCBL4 a gene, or overexpressing in said plant a TaCBL4 protein; The TaCBL4 gene encodes the TaCBL4 protein; The amino acid sequence of the TaCBL4 protein is shown as SEQ ID NO: 1; The plant is wheat; The pathogenic fungus of the stem base rot is Pseudocercosporella herpotrichoides Fusarium pseudograminearum .

2. The method of obtaining a plant resistant to stem base rot according to claim 1, characterized in that, The TaCBL4 The CDS sequence of the gene is shown as SEQ ID NO:

2.

3. The method of obtaining a plant resistant to stem base rot according to claim 1, characterized in that, Constructing a vector overexpressing the gene, transforming the vector into Agrobacterium, and infecting the plant with the Agrobacterium. TaCBL4 Constructing a vector overexpressing the gene, transforming the vector into Agrobacterium, and infecting the plant with the Agrobacterium.

4. The method of obtaining a plant resistant to stem base rot according to claim 3, characterized by, The upstream primer for constructing the vector is shown as SEQ ID NO: 3, and the downstream primer is shown as SEQ ID NO:

4.

5. TaCBL4 The use of the gene in the prevention and treatment of basal stem rot, characterized in that, Overexpression in plants TaCBL4 Genes, or overexpression of the TaCBL4 protein in plants; The TaCBL4 The gene encodes the TaCBL4 protein; The amino acid sequence of the TaCBL4 protein is shown as SEQ ID NO: 1; The plant is wheat; The pathogenic fungus of the stem base rot is Pseudocercosporella herpotrichoides Fusarium pseudograminearum .

6. The method of claim 5, wherein the method further comprises: TaCBL4 The application of the gene in the prevention and treatment of stem base rot, characterized in that, The TaCBL4 The CDS sequence of the gene is shown as SEQ ID NO: 2.

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